SUMMARY The majority of patients with myelodysplastic syndromes (MDS), a heterogeneous group of blood disorders characterized by ineffective and clonal hematopoiesis, carry a somatic mutation affecting an RNA splicing factor. The most commonly mutated splicing factor is SF3B1, a core component of the spliceosome that is preferentially mutated in MDS with ring sideroblasts (MDS-RS). Although SF3B1 mutations are among the most common genetic lesions in MDS, they are nonetheless relatively poorly understood. Our incomplete understanding of SF3B1 mutations is due in part to the absence of a model system that recapitulates hallmark disease phenotypes, including ring sideroblast formation and ineffective erythropoiesis. As a consequence, it is unclear how SF3B1 mutations alter RNA splicing mechanisms, which specific mis-spliced genes drive hallmark disease phenotypes, and whether SF3B1-mutant cells can be killed by targeted therapies. Here, we propose to elucidate the functional basis as well as mechanistic and phenotypic consequences of SF3B1 mutations in MDS-RS. Our team consists of a stem cell biologist with expertise in hematologic disease modeling (Doulatov), a basic scientist with expertise in RNA splicing and functional genomics (Bradley), and a physician-scientist with expertise in erythropoiesis and heme biology (Abkowitz). In preliminary studies, we generated MDS-RS patient-derived induced pluripotent stem cells (iPSCs) that recapitulate hallmark disease phenotypes during erythroid differentiation, identified specific mis-spliced genes that contribute to ineffective erythropoiesis, and performed functional genomic screens to identify molecular vulnerabilities of SF3B1-mutant cells. We propose to build on those preliminary studies as follows: Aim 1, Define the molecular consequences of SF3B1 mutations for mRNA splicing, stability, and translation; Aim 2, Determine the functional basis of ring sideroblast formation and ineffective erythropoiesis in SF3B1-mutant MDS-RS; Aim 3, Identify therapeutic opportunities for treating MDS-RS with SF3B1 mutations. The significance of these studies is that they will elucidate the mechanistic and functional consequences of SF3B1 mutations in MDS-RS. The health relatedness is that the proposed work may identify new opportunities for treating MDS by specifically targeting SF3B1-mutant cells. As the incidence of MDS is rising and patients with SF3B1-mutant MDS-RS face life-long transfusion burdens and associated morbidity and mortality, there is a public health need to develop new therapies for this disorder.